Near-infrared bioluminescent protein, gene thereof, protein pair, expression cassette, use method of expression cassette, cell, transgenic microorganism and kit

Abstract

The invention relates to a novel near-infrared bioluminescent protein based on a BRET principle, the near-infrared bioluminescent protein is formed by serially connecting and fusing a fluorescence activated protein NirF and a luciferase NLuc, and the fusion mode comprises NirF-N, N-NirF and NirF-N-NirF. According to the present invention, the near-infrared light with the wavelength of 680 nm can be emitted through the BRET by using the NLuc light-emitting substrate, such that the characteristics of low background signal, high BRET efficiency, strong near-infrared signal and the like are provided, and the ultra-high sensitivity is provided in the protein interaction detection. The invention also provides a gene of the near-infrared bioluminescent protein, a protein pair for bioluminescent resonance energy transfer detection, an expression box and a using method thereof, and cells, transgenic microorganisms and a kit for producing the near-infrared bioluminescent protein.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to BRET-type bioluminescent proteins in bioluminescence imaging technology. Background Art

[0002] Bioluminescent proteins have the characteristics of not requiring excitation light and having extremely low background signals, and have become one of the important means of in vivo optical imaging. At present, bioluminescence imaging technology has been widely used in research fields such as tracking cells in vivo, monitoring the transcription and post-transcriptional regulation of specific genes, medical diagnosis, and infectious diseases. However, luciferases derived from marine organisms have short emission wavelengths, and luciferases derived from insects have low quantum yields, which limits their wide application in living animals. Bioluminescence resonance energy transfer (BRET), that is, the phenomenon that energy is transferred from a bioluminescent donor to a receptor through non-radiative dipole-dipole coupling. BRET technology can combine a high-brightness short-wavelength luciferase and a fluorescent protein with a high fluorescence quantum yield to produce a high-brightness bioluminescent protein with a red-shifted wavelength.

[0003] In 1999, Xu et al. developed the BRET1 system for the study of circadian rhythms in cyanobacteria, using Renilla luciferase (Rluc) and EYFP as the donor and acceptor of BRET, respectively (Xu, Piston et al. 1999). In 2002, Bertrand et al. developed the BRET2 system using GFP as the acceptor (Bertrand, Parent et al. 2002). There was a good separation between the emission peaks of the BRET2 donor and acceptor, and its signal-to-noise ratio was significantly improved compared to BRET1. Pfleger et al. replaced Rluc with Rluc8 on the basis of BRET2 and obtained eBRET (extended BRET) with a signal intensity 5 times that of BRET2 (Pflegre, Dromey et al. 2006). Thereafter, researchers fused the Rluc8 mutant with the red fluorescent protein mOrange to develop BRET3 (De, Ray et al. 2009). The light output of BRET3 was redshifted (564 nm), and its photon intensity and spectral resolution were significantly improved. In 2012, Hall et al. optimized the structure of the small catalytic subunit of the luciferase from the deep-sea shrimp (Oplophorus gracilirostris) and obtained a new luciferase NanoLuc (NLuc) with a molecular weight of only 19 kDa (Hall, Unch et al. 2012). Due to its small molecular weight, strong luminescence signal, narrow bioluminescence spectrum and other characteristics, NLuc has become the first choice for protein fusion tags in a variety of applications. In 2015, Machleidt et al. developed a new NanoBRET system using NLuc as the donor and the HaloTag fusion tag as the acceptor. By selecting a red-light-emitting fluorophore (635 nm) attached to the HaloTag, an overall spectral separation of more than 175 nm was achieved, but its BRET efficiency was not high (Machleidt, Woodroofe et al. 2015). In 2016, the Michael team discovered a cyan light-excitable orange-red fluorescent protein CyOFP1, and then fused CyOFP1 with NLuc to obtain a new bioluminescence probe Antares (Chu, Oh et al. 2016). As a highly sensitive bioluminescence reporter gene, Antares can produce more stable and stronger signals in deep tissues, and the emission wavelength is shifted by 114 nm, but the emission wavelength of Antares is only 589 nm.

[0004] However, most of the emission wavelengths of the currently available BRET systems are concentrated below 600 nm, which is not conducive to cell and in vivo tissue imaging research. Therefore, there is an urgent need to develop near-infrared BRET probes with longer wavelengths and stronger signals to better solve the problem of in vivo deep tissue imaging. However, the luminescence intensity of the above systems is still not very ideal, which severely restricts the wide application of the above systems. The present invention combines a novel large Stokes shift near-infrared fluorescent activating protein with a highly bright luciferase to obtain a BRET system with ultra-high sensitivity, and also obtains a highly bright near-infrared bioluminescent protein. Summary of the Invention

[0005] In view of the deficiencies of the prior art, the present invention provides a highly bright near-infrared bioluminescent protein, which provides a new tool for in vivo deep tissue imaging of live animals and can also be used for highly sensitive protein interaction analysis. In a preferred implementation process, the near-infrared bioluminescent protein is obtained by the method of directed evolution.

[0006] To achieve the above technical objectives, the technical solutions provided by the present invention are as follows:

[0007] An embodiment of the present invention provides a near-infrared bioluminescent protein, which includes: (a) an amino acid sequence as shown in SEQ ID No: 1, 2, 3 or 4; or (b) the near-infrared bioluminescent protein has at least 90% identity with the amino acid sequence shown in SEQ ID No: 1, 2, 3 or 4.

[0008] In an embodiment of the present invention, the near-infrared bioluminescent protein has at least 92% identity with the amino acid sequence shown in SEQ ID No: 1, 2, 3 or 4.

[0009] In an embodiment of the present invention, the amino acid sequence of the near-infrared bioluminescent protein is as shown in SEQ ID No: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60.

[0010] In an embodiment of the present invention, the near-infrared bioluminescent protein includes a truncated mutant in which amino acid residues in the protein linking region are partially deleted.

[0011] Another embodiment of the present invention provides a gene of a near-infrared bioluminescent protein, and the protein encoded by the gene of the near-infrared bioluminescent protein is the above-mentioned near-infrared bioluminescent protein.

[0012] Another embodiment of the present invention provides a protein pair for bioluminescence resonance energy transfer detection. The protein pair is obtained by splitting the aforementioned near-infrared bioluminescent protein. Among them, the acceptor of bioluminescence resonance energy transfer is the fluorescent activation protein NirF or its mutant, and the donor is the corresponding luciferase NLuc or its mutant. The amino acid sequence of the fluorescent activation protein NirF is shown in SEQ ID NO: 61, and the amino acid sequence of the luciferase NLuc is shown in SEQ ID NO: 62.

[0013] Another embodiment of the present invention provides an expression cassette, which includes: the gene of the near-infrared bioluminescent protein as described above and a conditional component, wherein in a host cell, the gene of the near-infrared bioluminescent protein is expressed under the control of the conditional component.

[0014] Another embodiment of the present invention provides a method for using an expression cassette, which includes integrating the expression cassette as described above into the cell genome or introducing it into the cell as an extrachromosomal element, so as to be able to express the above-mentioned near-infrared bioluminescent protein.

[0015] Another embodiment of the present invention provides a cell for producing the above-mentioned near-infrared bioluminescent protein, and the cell contains the above-mentioned expression cassette.

[0016] Another embodiment of the present invention provides a transgenic microorganism, and the transgenic microorganism includes the gene of the near-infrared bioluminescent protein.

[0017] Another embodiment of the present invention provides a kit, which contains the near-infrared bioluminescent protein, or the gene of the near-infrared bioluminescent protein, or the expression cassette.

[0018] Advantages of the present invention:

[0019] (1) The present invention provides a near-infrared bioluminescent protein with high brightness, and its emission spectrum can reach 680 nm, which greatly improves the depth and sensitivity of in vivo imaging, effectively solves the problem of low brightness in the near-infrared band of existing bioluminescent proteins, and improves the performance of existing bioluminescent proteins in biological detection.

[0020] (2) The highly sensitive BRET pair provided by the present invention has the characteristics of almost no background signal in the near-infrared band and an extremely high dynamic range, greatly improving the sensitivity of the BRET pair and enhancing the performance of existing bioluminescence detection technologies. Description of the Drawings

[0021] Figure 1a-1b : NirFAP Fluorescence Spectra and Luminescence Spectra of Different Luciferases( Figure 1a ) and Schematic Diagram of the Prototype Structure of Near-Infrared Bioluminescent Protein( Figure 1b ).

[0022] Figure 2a-2b : Purified Protein of Near-Infrared Bioluminescent Protein Prototype( Figure 2a ) and Its Luminescence Spectrum in Living Cells( Figure 2b ).

[0023] Figure 3a-3l : Luminescence Spectra of Different Truncations, Heat Maps of Luminescence Intensity (Cyan) and Heat Maps of BRET Efficiency (Magenta).

[0024] Figure 4 : SDS-PAGE Analysis of Near-Infrared Bioluminescent Proteins NirNL and NirF(Δ1)-(Δ3)N Proteins

[0025] Figure 5 : Luminescence Spectra of N-NirF, NirNL and NirF(Δ1)-(Δ3)N and Their Prototype NirF-N Protein

[0026] Figure 6a-6d : Spectra of Near-Infrared Bioluminescent Protein NirNL and Its Prototype Protein in Different Mammalian Cell Lines

[0027] Figure 7a-7b : Luminescence Images of Different Bioluminescent Proteins in HEK293T Cells( Figure 7a ) and Quantitative Results( Figure 7b ).

[0028] Figure 8a-8b : Luminescence Images of Near-Infrared Bioluminescent Protein NirNL and Orange Bioluminescent Protein Antares in Mice( Figure 8a ) and Quantitative Results( Figure 8b ).

[0029] Figure 9a-9b : Schematic Diagram of the Principle for Detecting Drug-Induced Protein Interactions( Figure 9a ) and Test Results of Purified Proteins( Figure 9b ).

[0030] Figure 10a-10c : Luminescence Images of the Interaction between FRB and FKBP Induced by Rapamycin in HEK293T Cells( Figure 10a ) and the Corresponding Cell Luminescence Spectra( Figure 10b 、 Figure 10c ). Detailed Implementation Modes

[0031] The present invention will be further described below with reference to embodiments. These embodiments are only for illustrative purposes and do not constitute any limitation to the scope of the present invention. Conventional genetic engineering and molecular biology cloning methods are mainly used in the embodiments, and these methods are well-known to those of ordinary skill in the art. For example, relevant chapters in the "Molecular Biology Experiment Reference Manual" by Jane Roskams et al. Those of ordinary skill in the art can successfully implement the present invention according to the following embodiments and specific circumstances without difficulty.

[0032] The present invention provides a near-infrared bioluminescent protein, which is a bioluminescent protein composed of a fluorescent activation protein and a luciferase fused based on the BRET principle and can be used as a biological probe.

[0033] The present invention provides a BRET protein pair capable of being used for bioluminescence resonance energy transfer detection, and the BRET protein pair includes a luciferase mutant as a donor and a large Stokes shift near-infrared fluorescent activation protein as a receptor.

[0034] In some specific embodiments of the present invention, the bioluminescence resonance energy transfer receptor is selected from the fluorescent activation protein NirFAP (or simply NirF) or a truncated mutant with partial deletion of its amino acid residues (i.e., a truncated body or a truncated mutant), which emits fluorescence at 680 nm. The bioluminescence resonance energy transfer donor is selected from the luciferase NLuc (or NanoLuc, or simply N) or its truncated variant. The connection mode between NirFAP and NLuc is a direct tandem, and the connection modes are NirF-N or N-NirF or NirF-N-NirF.

[0035] The present invention provides a near-infrared bioluminescent protein, which is formed by reacting and connecting the above-mentioned protein pair for bioluminescence resonance energy transfer detection, and it is a fusion protein having an amino acid sequence selected from the following:

[0036] (1) The amino acid sequences of the fluorescent activation protein (Acceptor, A) and the luciferase (Donor, D) in series to form A-D, D-A, and A-D-A structures, and (2) truncated bodies having at least 90%, 92%, 94%, 96%, 98%, 99% sequence identity with (1) and having near-infrared luminescence characteristics, which can be, for example, amino acid sequences with 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

[0037] In one embodiment, the near-infrared bioluminescent protein has the sequence shown in SEQ ID No: 1 (MSRAAQLLPGTWQVTMTQPDGFTSQGQMHFQPRSPYTMDVVAQGTISDGRPISGYGKVTVKTPDTLDVDITYPSLGNIKAQGQITMDSPTQFKFDATTKGAGNFTGRLTGTLQRQEMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILA), or a truncated form thereof having at least 90%, 92%, 94%, 96%, 98%, 99% sequence identity and retaining the near-infrared luminescence property, which can be, for example, an amino acid sequence with 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

[0038] In one embodiment, the near-infrared bioluminescent protein has the sequence shown in SEQ ID No: 3 (MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAMSRAAQLLPGTWQVTMTQPDGFTSQGQMHFQPRSPYTMDVVAQGTISDGRPISGYGKVTVKTPDTLDVDITYPSLGNIKAQGQITMDSPTQFKFDATTKGAGNFTGRLTGTLQRQE), or a truncated form thereof having at least 90%, 92%, 94%, 96%, 98%, 99% sequence identity and retaining the near-infrared luminescence property, which can be, for example, an amino acid sequence with 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

[0039] In some embodiments, when the C-terminus of the fluorescent activation protein is linked to the N-terminus of the luciferase, the truncated amino acids are selected from the amino acids at positions 112-115 of the fluorescent activation protein and / or the amino acids at positions 1-5 of the luciferase.

[0040] When the C-terminus of luciferase is linked to the N-terminus of a fluorescence activation protein, the truncated amino acids are selected from the amino acids at positions 167-171 of luciferase and / or the amino acids at positions 1-5 of the fluorescence activation protein.

[0041] In one embodiment, when the fluorescence activation protein is NirF and the luciferase is NLuc, the fusion proteins are selected from one of the following combinations: NirF(Δ0)-(Δ0)N, NirF(Δ0)-(Δ1)N, NirF(Δ0)-(Δ2)N, NirF(Δ0)-(Δ3)N, NirF(Δ0)-(Δ4)N, NirF(Δ0)-(Δ5)N, NirF(Δ1)-(Δ0)N, NirF(Δ1)-(Δ1)N, NirF(Δ1)-(Δ2)N, NirF(Δ1)-(Δ3)N, NirF(Δ1)-(Δ4)N, NirF(Δ1)-(Δ5)N, NirF(Δ2)-(Δ0)N, NirF(Δ2)-(Δ1)N, NirF(Δ2)-(Δ2)N, NirF(Δ2)-(Δ3)N, NirF(Δ2)-(Δ4)N, NirF(Δ2)-(Δ5)N, NirF(Δ3)-(Δ0)N, NirF(Δ3)-(Δ1)N, NirF(Δ3)-(Δ2)N, NirF(Δ3)-(Δ3)N, NirF(Δ3)-(Δ4)N, NirF(Δ3)-(Δ5)N, NirF(Δ4)-(Δ0)N, NirF(Δ4)-(Δ1)N, NirF(Δ4)-(Δ2)N, NirF(Δ4)-(Δ3)N, NirF(Δ4)-(Δ4)N, NirF(Δ4)-(Δ5)N. Here, the numbers in the parentheses refer to the number of truncated amino acids included in the fragments of luciferase and the fluorescence activation protein.

[0042] In one embodiment, when the fluorescent activation protein is NirF and the luciferase is NLuc, the fusion protein is selected from one of the following combinations: N(Δ0)-(Δ0)NirF, N(Δ0)-(Δ1)NirF, N(Δ0)-(Δ2)NirF, N(Δ0)-(Δ3)NirF, N(Δ0)-(Δ4)NirF, N(Δ0)-(Δ5)NirF, N(Δ1)-(Δ0)NirF, N(Δ1)-(Δ1)NirF, N(Δ1)-(Δ2)NirF, N(Δ1)-(Δ3)NirF, N(Δ1)-(Δ4)NirF, N(Δ1)-(Δ5)NirF, N(Δ2)-(Δ0)NirF, N(Δ2)-(Δ1)NirF, N(Δ2)-(Δ2)NirF, N(Δ2)-(Δ3)NirF, N(Δ2)-(Δ4)NirF, N(Δ2)-(Δ5)NirF, N(Δ3)-(Δ0)NirF, N(Δ3)-(Δ1)NirF, N(Δ3)-(Δ2)NirF, N(Δ3)-(Δ3)NirF, N(Δ3)-(Δ4)NirF, N(Δ3)-(Δ5)NirF, N(Δ4)-(Δ0)NirF, N(Δ4)-(Δ1)NirF, N(Δ4)-(Δ2)NirF, N(Δ4)-(Δ3)NirF, N(Δ4)-(Δ4)NirF, N(Δ4)-(Δ5)NirF. Wherein, the numbers in the brackets refer to the number of truncated amino acids included in the luciferase and the fluorescent activation protein fragment.

[0043] In one embodiment, when the fluorescent activation protein is NirF and the luciferase is NLuc, the fusion protein is selected from one of the following combinations: NirF(Δ2)-(Δ2)N-NirF, NirF(Δ1)-(Δ3)N NirF, where the numbers in the brackets refer to the number of truncated amino acids included in the luciferase and the fluorescent activation protein fragment.

[0044] The amino acid sequence of the fluorescent activation protein NirF is shown in SEQ ID No: 61:

[0045]

[0046] The amino acid sequence of the luciferase NLuc is shown in SEQ ID No: 62:

[0047]

[0048] In some embodiments, the near-infrared bioluminescent protein has the amino acid sequence shown in Table 1 below.

[0049] Table 1: Amino acid sequence table of near-infrared bioluminescent protein

[0050]

[0051]

[0052]

[0053]

[0054]

[0055]

[0056]

[0057]

[0058]

[0059]

[0060] The present invention also provides a gene (nucleic acid molecule) of a near-infrared bioluminescent protein, which comprises: (a): the coding sequence of the near-infrared bioluminescent protein described in any embodiment herein, or (b): the complementary sequence of (a), or (c): a fragment of (a) or (b) reasonably split at the linking region.

[0061] The present invention also relates to variants of the above nucleic acid molecules, including nucleic acid sequences or their complementary sequences encoding genes, fragments, analogs, derivatives, soluble fragments and variants of the near-infrared bioluminescent protein of the present invention.

[0062] On the other hand, the present invention also provides a nucleic acid construct comprising the nucleic acid molecule described herein. This nucleic acid sequence encodes the fusion protein described in the present invention.

[0063] In some embodiments, the nucleic acid construct is a cloning vector, an expression vector or a recombinant vector.

[0064] In some embodiments, the nucleic acid molecule is operably linked to an expression control sequence.

[0065] In some embodiments, the expression vector is selected from prokaryotic expression vectors, eukaryotic expression vectors and viral vectors.

[0066] On the other hand, the present invention also provides a host cell, which: (1) expresses the fusion protein described in any embodiment of the present invention; (2) contains the nucleic acid molecule described in any embodiment of the present invention; or (3) contains the nucleic acid construct described in any embodiment of the present invention. The host cell is preferably Escherichia coli.

[0067] On the other hand, the present invention provides a method for preparing the near-infrared luminescent bioprotein described herein, comprising: providing a host cell expressing the near-infrared luminescence described herein, culturing the host cell under conditions for expression in the cell, and isolating the near-infrared luminescent probe.

[0068] In one or more embodiments, the method comprises the following steps: 1) incorporating a nucleic acid molecule encoding the near-infrared luminescent probe described herein into an expression vector; 2) transferring the expression vector into a host cell; 3) culturing the host cell under conditions suitable for the expression of the expression vector, and 4) isolating the near-infrared luminescent probe.

[0069] The highly bright near-infrared bioluminescent protein obtained by the present invention can be used as a luminescent probe, which has high sensitivity and significantly improved luminescence intensity in the infrared band, can overcome the disadvantages of the previous BRET-based bioluminescence system, can achieve highly sensitive detection of protein-protein interactions, and can efficiently complete luminescence imaging in living animals and cells.

[0070] The pEGFP-C1 plasmid vector used in the examples was purchased from Invitrogen, the pLVX-puro plasmid vector was purchased from Clontech, and the pCDFDuet-1 plasmid vector was purchased from Novagen. All primers used for PCR were synthesized, purified and correctly identified by mass spectrometry by Shanghai Jerry Biotechnology Co., Ltd. The expression plasmids constructed in the examples were all subjected to sequence determination, which was completed by Jie Li Sequencing Company. The PrimeSTAR DNA polymerase used in the PCR reactions in each example was purchased from TaKaRa. Restriction endonucleases such as BamHI, XhoI, and Acc65I were purchased from Fermentas. The Hieff Clone TM One Step cloning kit was purchased from Shanghai Yisheng Biotechnology Co., Ltd. Inorganic salt chemical reagents were all purchased from Shanghai Chemical Reagent Company, Sinopharm Group. Kanamycin and Streptomycin were both purchased from Ameresco. The 96-well white plates were purchased from Grenier.

[0071] The BL21(DE3) strain used in the examples was purchased from Invitrogen. HeLa cells, HEK293T cells, H1299 cells and A549 cells were purchased from the Cell Bank of the Chinese Academy of Sciences' Committee for Type Culture Collection. ICR mice were purchased from Shanghai Jiesijie Experimental Animal Co., Ltd. The DNA purification kit used in the examples was purchased from BBI (Canada), and the ordinary plasmid miniprep kit was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd. The main instruments used in the examples: Synergy Neo2 multimode microplate reader (Bio-Tek, USA), Microfuge22R tabletop high-speed refrigerated centrifuge (Beckman, USA), PCR amplifier (Biometra, Germany), IVIS Spectrum CT in vivo imaging system (PerkinElmer, USA), nucleic acid electrophoresis apparatus (Shenneng Bojin Co., Ltd.).

[0072] The meanings of the abbreviations are as follows: "h" refers to hour, "min" refers to minute, "s" refers to second, "d" refers to day, "μL" refers to microliter, "mL" refers to milliliter, "L" refers to liter, "bp" refers to base pair, "mM" refers to millimole, "μM" refers to micromole.

[0073] Experimental methods and materials in the examples:

[0074] Plasmid construction:

[0075] 1. Preparation of linearized vector: Select appropriate cloning sites and prepare linearized vectors by restriction enzyme digestion.

[0076] 2. Preparation of insertion fragments by PCR amplification: By introducing 15 bp (excluding restriction enzyme sites) of linearized vector terminal homologous sequences at the 5' ends of the forward and reverse PCR primers of the insertion fragments, the 5' and 3' ends of the insertion fragment PCR products respectively carry exactly the same sequences corresponding to the two ends of the linearized vector.

[0077] 3. Determination of the concentrations of linearized vectors and insertion fragments: Use a Synergy Neo2 multimode microplate reader to determine the DNA concentration obtained by purification.

[0078] 4. Recombination reaction: The amount of vector used in the recombination reaction system is 0.03 pmol; the optimal molar ratio of vector to insertion fragment is 1:2 - 1:3, that is, the optimal amount of insertion fragment used is 0.06 - 0.09 pmol. After the system is prepared, mix each component and place it at 50 °C for reaction for 20 min. When the insertion fragment > 5 kb, the incubation temperature can be extended to 40 min. After the reaction is completed, immediately perform transformation. Subsequently, positive clones are identified by colony PCR and sequencing.

[0079] Protein purification:

[0080] The correctly sequenced plasmid was transformed into BL21(DE3). After single colonies grew out, the single colonies were picked into test tubes for primary culture. After overnight culture, the bacterial solution was inoculated into LB medium at a ratio of 1:100 and cultured on a shaker at 37°C. When the OD 600 reached 0.6, IPTG (final concentration 1 mM) was added to induce protein expression, and the expression was carried out on a shaker at 16°C for 24 - 36 h. After the expression ended, the cells were collected by centrifugation, the cell pellet was resuspended in Buffer, lysed by sonication, and the protein was purified by affinity chromatography.

[0081] Mammalian cell culture and transfection:

[0082] The cells in this example were all cultured in a CO2 incubator with high - glucose medium (DMEM) containing 10% fetal bovine serum (FBS), streptomycin, and penicillin. When the growth reached 50 - 60% confluence, the cells were passaged. For transfection, HieffTrans TM (purchased from Yensen) was operated according to the instructions.

[0083] Luminescence test:

[0084] After the purified protein was identified by SDS - PAGE, the probe was diluted with assay buffer (100 mM HEPES, 100 mM NaCl, pH 7.4) to a protein solution with a final concentration of 0.2 - 5 μM. Furimazine was prepared into a stock solution with a final concentration of 100 μM with assay buffer (100 mM HEPES, 100 mM NaCl, pH 7.4), and the ligand of NirFAP was prepared into a stock solution with a final concentration of 1 mM with the same buffer. Take 50 μL of 1 μM protein solution, add an appropriate amount of ligand to make its final concentration 5 μM, incubate at 37°C for 10 minutes, then add 50 μL of 100 μM furimazine, and immediately perform luminescence spectral scanning, which is completed using a multifunctional microplate reader. Mammalian cells were cultured with DMEM medium, plated and transfected in a 96 - well white plate. Each well of the adherent cells that had completed expression was replaced with 50 μL of medium without phenol red containing 10 μM ligand, incubated in a cell culture incubator at 37°C and 5% CO2 for 1 h, then 50 μL of 100 μM furimazine was added, and the detection was immediately carried out on a live imaging instrument or a multifunctional microplate reader.

[0085] Example 1: Synthesis of a near - infrared bioluminescent protein prototype expression vector

[0086] Select the commonly used luciferase NLuc or the luciferase QLuc whose emission spectrum highly overlaps with the excitation spectrum of NirF as the BRET donor ( Figure 1a)。The NirFAP and NLuc or QLuc genes were amplified by PCR, and fusion proteins with different fusion orders were constructed by overlap extension PCR ( Figure 1b ) gene. Further, the obtained fusion genes were cloned into the pCDFDuet-1 vector and the pEGFP-C1 vector. The vectors and fragments were ligated by homologous recombination, and the products were transformed into competent cells, then spread on LB plates containing antibiotics and incubated overnight at 37°C. Single colonies on the plates were picked for colony PCR. The positive clones obtained by colony PCR were inoculated into 5 mL of LB medium and cultured overnight, and then the plasmids were extracted for sequencing identification. After correct sequencing, they could be tested.

[0087] Example 2: Preliminary testing of prototype proteins obtained by fusing NirF with different luciferases NLuc and QLuc and their preliminary testing in mammalian cells

[0088] The near-infrared bioluminescent prototype proteins were diluted to 0.2 μM using a test buffer (100 mM HEPES, 100 mM NaCl, pH 7.4), and furimazine was prepared as a stock solution with a final concentration of 50 μM. The ligand of NirF was prepared as a stock solution with a final concentration of 1 mM using the same buffer. 50 μL of a 1 μM protein solution was taken, and an appropriate amount of ligand was added to make its final concentration 5 μM. It was incubated at 37°C for 10 minutes, and then 50 μL of 25 μM furimazine was added. Immediately, the luminescence spectrum was measured using a multimode microplate reader. The test results showed that Q-NirF and NirF-Q had higher BRET efficiencies, while N-NirF and NirF-N had lower corresponding BRET efficiencies, but the luminescence intensity in the infrared region was at the same level as that of Q-NirF and NirF-Q. After that, we conducted preliminary tests on the prototype proteins expressed in mammalian cells. The test results showed that the luminescence intensities of Q-NirF and NirF-Q in living cells were very low, while N-NirF and NirF-N had higher luminescence intensities in the infrared region. This indicates that N-NirF and NirF-N have better application values than QLuc-based BRET proteins in living cells.

[0089] Example 3: Construction, expression and testing of different near-infrared bioluminescent protein prototype truncations

[0090] In this embodiment, luciferase NLuc was ligated based on pCDFDuet-1-NirF, and the following amino acids were selected for truncation to obtain the corresponding pCDFDuet-1-NirF(ΔX)-(ΔY)N and pCDFDuet-1-N(ΔX)-(ΔY)NirF plasmids, where X and Y represent truncation at the N-terminus and C-terminus of NirF and NLuc, with X = 0, 1, 2, 3, 4 and Y = 0, 1, 2, 3, 4, 5. As an exemplary display, the NirF-N nucleic acid sequence is as shown in SEQ ID No:1.

[0091] The DNA fragment of NLuc was amplified by PCR, and at the same time, the NLuc terminal homologous sequence was introduced at the 5'-end of the primer. The PCR amplification produced a linearized pCDFDuet-1-NirF vector, with the 5'- and 3'-terminal ends respectively carrying exactly the same sequences (15 bp - 20 bp) corresponding to the two terminal ends of NLuc. The linearized pCDFDuet-1-NirF and NLuc fragments underwent homologous recombination under the action of infusion. The product was transformed into DH5α, and the transformed DH5α was spread on an LB plate with antibiotics (streptomycin 100 μg / mL) and incubated overnight at 37°C. The positive clones identified by colony PCR were extracted with plasmids and then sequenced. The sequencing was completed by Jie Li Sequencing Company. After correct sequencing, the recombinant plasmid was transformed into BL21(DE3) for induced expression, and the protein was purified. The size was around 33 kDa by SDS-PAGE electrophoresis. This size corresponded to that of the fusion protein NirF-N containing the His-tag purification tag expressed by pCDFDuet-1-NirF-N.

[0092] The luminescence spectrum was measured in Escherichia coli expressing NirF(ΔX)-(ΔY)N and N(ΔX)-(ΔY)NirF fusion proteins. 50 μL of the bacterial solution with OD600 = 0.1 was taken, and a ligand with a final concentration of 1 μM was added. After incubation at 37°C for 10 minutes, 10 μL of 100 μM furimazine was added, and the luminescence spectrum was scanned using a multifunctional microplate reader. All the sequences listed in Table 1 can be used as the near-infrared luminescent proteins of the present invention, where Figure 3a corresponding to SEQ ID No: 5 - 9, Figure 3b corresponding to SEQ ID No: 2, 10 - 14, Figure 3c corresponding to SEQ ID No: 15 - 19, Figure 3d corresponding to SEQ ID No: 20 - 25, Figure 3e corresponding to SEQ ID No: 26 - 31, Figure 3f corresponding to SEQ ID No: 32 - 36, Figure 3g corresponding to SEQ ID No: 37 - 42, Figure 3hCorresponding to SEQ ID No: 43 - 48, Figure 3i Corresponding to SEQ ID No: 49 - 54, Figure 3j Corresponding to SEQ ID No: 55 - 60. In addition, Figure 3a -j all contain SEQ ID No: 1 and 4 as controls. The detection results show that BRET effects occurred in all NirF(ΔX)-(ΔY)N expressed in Escherichia coli; as Figure 3f to 3j , the detection results show that BRET effects occurred in all N(ΔX)-(ΔY)NirF expressed in Escherichia coli.

[0093] To quantitatively test the BRET efficiency of different truncated mutations, further identification was carried out in the Escherichia coli lysates expressing NirFAP(ΔX)-(ΔY)NLuc and NLuc(ΔX)-(ΔY)NirFAP fusion proteins. Take 50 μL of the supernatant solution, add a ligand with a final concentration of 5 μM, incubate at 37 °C for 10 minutes, then add 50 μL of 100 μM furimazine, and perform luminescence spectral scanning using a multi-functional microplate reader. As Figure 3k and 3l shown, the detection results show that in the disrupted supernatant expressing NirF-N fusion protein, those with a BRET efficiency greater than 0.8 are NirF(Δ2)-(Δ2)N, NirF(Δ1)-(Δ3)N, and NirF(Δ0)-(Δ4)N.

[0094] Example 4: Purification of Preferred Near-Infrared Bioluminescent Proteins

[0095] To determine the efficacy of the obtained near-infrared bioluminescent protein, plasmids expressing different mutants MSRAAQLLPGTWQVTMTQPDGFTSQGQMHFQPRSPYTMDVVAQGTISDGRPISGYGKVTVKTPDTLDVDITYPSLGNIKAQGQITMDSPTQFKFDATTKGAGNFTGRLTGTLQRQTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILA (SEQ ID No:2) and MSRAAQLLPGTWQVTMTQPDGFTSQGQMHFQPRSPYTMDVVAQGTISDGRPISGYGKVTVKTPDTLDVDITYPSLGNIKAQGQITMDSPTQFKFDATTKGAGNFTGRLTGTLQRFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILA (SEQ ID No:4) were transformed into BL21(DE3) for protein expression. The transformed colonies were picked and cultured overnight in test tubes. Then, 1 mL of the bacterial solution was inoculated into 100 mL of LB medium at a ratio of 1%, and cultured on a shaker at 37°C and 220 rpm. When the OD600 value of the bacterial solution reached 0.6, IPTG (final concentration 1 mM) was added to induce protein expression, and the induction was carried out at 18°C for 24 - 48 h. After the expression ended, the bacterial cells were collected by centrifugation, resuspended in an appropriate amount of His Buffer A, lysed by sonication, centrifuged at 12,000 rpm at 4°C for 20 min, and the protein supernatant was added to the affinity column in batches; 5 column volumes of a washing buffer containing 50 mM imidazole were added to remove the vast majority of impurities; then, the target protein was eluted with an elution buffer containing 300 mM imidazole, and the target protein was collected according to the Bradford colorimetric process, labeled, and stored on ice for later use; the concentration of the purified protein was determined by the BCA method for later use. After determining the concentration of the purified protein, the size and purity of the purified protein were verified by SDS-PAGE. Figure 4To purify the electrophoretograms of different near-infrared luminescent proteins, the electrophoresis results showed that there was a protein slightly larger than 33 kDa, which was consistent with the size of the near-infrared bioluminescent protein of 34 kDa and met the expectations. On the other hand, Figure 4 it also showed that the purity of different near-infrared bioluminescent proteins was very high, and there were basically no contaminating proteins.

[0096] Example 5: Testing of Preferred Near-Infrared Bioluminescent Proteins

[0097] After the purified protein was identified by SDS-PAGE, it was diluted with the assay buffer (100 mM HEPES, 100 mM NaCl, pH 7.4) to a protein solution with a final concentration of 0.2 μM. Furimazine was formulated into a stock solution with a final concentration of 100 μM with the assay buffer (100 mM HEPES, 100 mM NaCl, pH 7.4), and the ligand of NirFAP was formulated into a stock solution with a final concentration of 1 mM with the same buffer. Take 50 μL of the 1 μM protein solution, add an appropriate amount of ligand to make its final concentration 5 μM, incubate at 37 °C for 10 minutes, then add 50 μL of 100 μM furimazine, and immediately perform luminescence spectral scanning, which was completed using a multifunctional microplate reader. The results are as Figure 5 shown. The BRET efficiency was greatly improved, and the near-infrared luminescence intensities of NirNL and NirF(Δ1)-(Δ3)N were 7 times and 5.6 times that of the prototype NirF-N, respectively.

[0098] Example 6: Spectra of Preferred Near-Infrared Bioluminescent Proteins in Different Mammalian Cells

[0099] In this example, we used a 96-well white plate for testing. The plasmids of cytosolic-expressed NLuc, N-NirF, NirF(Δ2)-(Δ2)N (NirNL), NirF(Δ1)-(Δ3)N, and Antares were transfected into HEK293T, A549, HeLa, and H1299 cells. The medium in the well plates of the living cells expressing the above proteins was replaced with 50 μL of medium without phenol red containing 10 μM NirFAP ligand, and incubated in a cell culture incubator at 37 °C and 5% CO2 for 1 h. Then, 50 μL of 50 μM furimazine was added, and the luminescence spectra of the proteins expressed in the living cells were tested using a microplate reader. As Figure 6a shown in a, b, c, and d, NirNL had the strongest near-infrared luminescence intensity in different mammalian cells (HEK293T, A549, HeLa, and H1299), indicating that the near-infrared bioluminescent proteins introduced in the present invention can be applied in different mammalian cell lines.

[0100] Example 7: Imaging of preferred near-infrared bioluminescent proteins in mammalian cells

[0101] In this example, we used a 96-well white plate for testing. The culture medium of HEK293T cells expressing Antares, NLuc, N-NirF, NirF(Δ1)-(Δ3)N, NirF(Δ2)-(Δ2)N (NirNL), and Antares was replaced with 50 μL of phenol red-free medium containing 10 μM ligand, incubated in a cell culture incubator at 37 °C and 5% CO2 for 1 h, and then 50 μL of 100 μM furimazine was added, and images were taken with a live imaging instrument. As Figure 7a and 7b shown, NirNL has the highest luminescence intensity in the near-infrared band.

[0102] Example 8: Comparison of the bioluminescence intensities of the preferred near-infrared bioluminescent proteins NirNL and Antares in live mice

[0103] In this example, we used the hydrodynamic method to inject 10 mg of plasmid into the tail vein of mice to express NirNL and Antares in the mouse liver respectively. After 24 hours of expression, 1 mmol of fluorescent activator protein ligand was injected intraperitoneally and then 1 mmol of furimazine was injected intravenously. Subsequently, images were taken with an IVIS Spectrum CT (PerkinElmer) live imaging instrument, and band-pass filters of 600 / 20 nm and 680 / 20 nm were used respectively. The test results showed that the full-spectrum luminescence intensity of NirNL was 2.0 times that of Antares, and the luminescence intensity in the infrared band (680 / 20) was 4.3 times that of Antares ( Figure 8a and 8b ). This indicates that the near-infrared bioluminescent protein NirNL introduced in the present invention has a higher luminescence intensity in live mice than existing BRET-type bioluminescent proteins.

[0104] Example 9: Detection of protein-protein interaction

[0105] In this example, we fused mTOR (FRB) and FK506 binding protein (FKBP) to the C-terminus of NirFAP and the N-terminus of NLuc respectively, constructed prokaryotic expression vectors of NirFAP-FRB and FKBP-Nluc and purified the proteins. Then, the two proteins with a final concentration of 100 nM were mixed, 2 mM fluorescent ligand and 100 nM rapamycin were added and incubated together, and finally, furimazine with a final concentration of 25 mM was added for reaction, and the emission spectrum was measured using a microplate reader. The test results showed that the luminescence intensity at 680 nm increased significantly after the addition of rapamycin (Figure 9b ) This indicates that the BRET pair introduced in the present invention can sensitively indicate the protein interaction between FRB and FKBP induced by rapamycin.

[0106] Example 10: Testing of drug-induced protein interaction in living cells

[0107] In this example, we constructed NirFAP-FRB and FKBP-Nluc into mammalian cell expression vectors. Then the two plasmids were co-transfected into HEK293T cells, and at the same time, the plasmid co-expressing CyOFP-FRB and FKBP-Nluc was co-transfected for comparison. After 24 h of transfection, a living imager and a microplate reader were used for detection respectively. The images taken by the living imager showed that the signal corresponding to 680 / 20 nm in the cells expressing NirFAP-FRB and FKBP-Nluc increased significantly after the addition of rapamycin; in contrast, the signal corresponding to 580 / 20 nm in the cells expressing CyOFP-FRB and FKBP-Nluc increased significantly after the addition of rapamycin ( Figure 10a ). The test results of the microplate reader showed that the signal corresponding to 680 nm in the cells expressing NirFAP-FRB and FKBP-Nluc increased by 310 times ( Figure 10b ), in contrast, the signal corresponding to 580 nm in the cells expressing CyOFP-FRB and FKBP-Nluc did not change after the addition of rapamycin ( Figure 10c ). This indicates that the BRET pair introduced in the present invention has higher sensitivity than the existing BRET pairs.

[0108] It should be understood that the dosages, reaction conditions, etc. in the examples of this specification are approximate values unless otherwise specified, and can be slightly changed according to the actual situation to obtain similar results. Unless otherwise specifically defined, all professional and scientific terms used herein have the same meaning as understood by those skilled in the art. All the documents mentioned herein are incorporated herein by reference. The preferred embodiments described in this specification are for illustrative purposes. Those skilled in the art can implement the present invention using methods and materials similar to those described herein to obtain the same or similar results. Any modifications or changes made to the present invention still fall within the scope defined by the appended claims of this application.

Claims

1. A near-infrared bioluminescent protein, characterized in that: The near-infrared bioluminescent protein includes: (a) an amino acid sequence as shown in SEQ ID No: 1, 2, 3 or 4; or (b) the amino acid sequence of the near-infrared bioluminescent protein has at least 90% identity with the amino acid sequence shown in SEQ ID No: 1, 2, 3 or 4.

2. The near-infrared bioluminescent protein according to claim 1, wherein: The amino acid sequence of the near-infrared bioluminescent protein is as shown in SEQ ID No: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60.

3. The near-infrared bioluminescent protein according to claim 1, wherein: The near-infrared bioluminescent protein includes a truncated mutant in which amino acid residues in the protein linking region are partially deleted.

4. A gene of a near-infrared bioluminescent protein, characterized in that: The protein encoded by the gene of the near-infrared bioluminescent protein is the near-infrared bioluminescent protein described in claim 1.

5. A protein pair for bioluminescence resonance energy transfer detection, characterized in that: The protein pair is obtained by splitting the near-infrared bioluminescent protein described in claim 1, wherein the acceptor of bioluminescence resonance energy transfer is the fluorescent activation protein NirF or its mutant, and the donor is the luciferase NLuc or its mutant; wherein the amino acid sequence of the fluorescent activation protein NirF is as shown in SEQ ID NO: 61, and the amino acid sequence of the luciferase NLuc is as shown in SEQ ID NO:

62.

6. An expression cassette, characterized in that: The expression cassette includes: the gene of the near-infrared bioluminescent protein as described in claim 4 and a conditional component, wherein the gene of the near-infrared bioluminescent protein as described in claim 4 is expressed under the control of the conditional component in a host cell.

7. A method for using an expression cassette, characterized in that: The expression cassette as described in claim 6 is integrated into the cell genome or introduced into the cell as an extrachromosomal element, so as to express the near-infrared bioluminescent protein as described in claim 1.

8. A cell that produces the near-infrared bioluminescent protein as described in claim 1, characterized in that: The cell contains the expression cassette as described in claim 6.

9. A transgenic microorganism, characterized in that, The transgenic microorganism includes the gene of the near-infrared bioluminescent protein as described in claim 4.

10. A kit, characterized in that: Comprising the near-infrared bioluminescent protein described in claim 1, or the gene of the near-infrared bioluminescent protein described in claim 4, or the expression cassette described in claim 6.

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